Evanescent Wave-imaging of GFP Constructs in the Membrane Adhesion Zone of Fibroblasts
A new design for an evanescent wave microscope was implemented to excite and monitor fluorescent molecules near the glass–water interface of an accessible cell chamber ( A and Materials and Methods). This imaging system included the capability to use low as well as high magnification objectives and to remotely scan the sample using micromotors. Evanescent wave microscopy, also known as total internal reflection fluorescence microscopy, has been used previously to study cell adhesion, binding to surfaces, and membrane trafficking (Axelrod 1981
; Thompson and Lagerholm 1997
; Burmeister et al. 1998
; Schmoranzer et al. 2000
; Toomre et al. 2000
). As a novel application, we reasoned that such a microscope could be used to monitor translocation of GFP-tagged signaling proteins to or from the plasma membrane within the contact area of living, adherent cells. Our experimental setup also allowed for parallel epifluorescence excitation. B shows unstimulated, GFP-transfected NIH 3T3 fibroblasts excited sequentially by epifluorescence and evanescent wave illumination. With the latter, only the adherent “footprint” of a cell is visualized. The high spatial resolution in the z direction results from a very short penetration depth of the excitation field (nominally ~70 nm). Since the illuminated sample is then effectively two-dimensional, the resolution in the x-y direction also improves and is set by the diffraction limit of light.
Figure 1 Evanescent wave imaging of PDGF-induced GFP–AktPH plasma membrane translocation in living fibroblasts. (A) Schematic view of the modified evanescent wave microscope design (see Materials and Methods for details). In short, the microscope uses (more ...)
Compared with confocal microscopy, the selective excitation and higher spatial resolution of evanescent wave microscopy make it better suited for measuring plasma membrane recruitment of signaling proteins. The sensitivity of the technique was assessed by comparing the relative excitation of a membrane-targeted GFP (using the palmitoylation–myristoylation sequence from Lyn) to that of soluble GFP ( C). Evanescent wave microscopy detects membrane localization simply as an elevated fluorescence intensity, allowing many cells to be observed at low magnification, as shown in C. After normalization by the epifluorescence signal for each cell (data not shown), it was determined that excitation of the membrane-targeted GFP was ~12-fold higher than for soluble GFP. The dynamic range of the assay for the relatively thin fibroblasts is therefore estimated to be over an order of magnitude.
GFP-labeled PH domains have been employed as quantitative molecular sensors to measure the local concentration of PI lipids in living cells over time (Stauffer et al. 1998
). Several pleckstrin homology (PH) domains bind specifically to the 3′ PIs PI(3,4,5)P3
(Lemmon et al. 1996
; Czech 2000
), and the GFP-conjugated PH domain of Akt has been used effectively as a fluorescent biosensor for 3′ PI lipids (Kontos et al. 1998
; Watton and Downward 1999
; Meili et al. 1999
; Servant et al. 2000
). D shows the increase in surface-proximal fluorescence intensity observed for NIH 3T3 fibroblasts, transfected with GFP–AktPH, after stimulation with PDGF at room temperature (~25°C). Stimulated translocation of this probe was robust and easily measured by evanescent wave microscopy. The PI 3-kinase inhibitors LY294002 (100 μM, D) or wortmannin (100 nM, not shown), but not vehicle alone, completely abolished GFP–AktPH translocation in our assay, suggesting that this probe binds specifically to 3′ PIs.
Evanescent Wave Microscopy Is Suitable for Monitoring Signal Transduction Events at Physiological Agonist Concentrations
Representative kinetic traces of PDGF-stimulated GFP–AktPH translocation are shown in E, showing minimal, intermediate, and maximal translocation responses. In accord with previous studies, maximal translocation was long-lived on the time scale of minutes (Watton and Downward 1999
). The dose response curve indicates that half-maximal translocation is achieved at a PDGF concentration yielding roughly half-maximal receptor binding (~0.2 nM; F). This is consistent with the absence of major bottlenecks between receptor binding and probe translocation, such as significant depletion of GFP–AktPH from the cytosol at submaximal PDGF doses. The slower translocation kinetics at intermediate doses ( E) is also consistent with receptor binding of PDGF being the rate-limiting step.
Although the affinities between PH domains and PI lipids measured in vitro are relatively low, it is still conceivable that most of the produced 3′ PI molecules are sequestered by the GFP–AktPH domains, thereby mediating a dominant-negative effect. If this were the case, the fold increase in surface-proximal fluorescence would be a decreasing function of the probe expression level. However, while the basal fluorescence varied over two decades of expression, the maximum fold increase in fluorescence intensity varied over a much smaller range, with no apparent reduction for all but the highest expressers ( G). This is consistent with most 3′ PIs remaining available for binding cellular targets except in cells that express the highest concentrations of GFP–AktPH domains. Thus, we do not attribute the observed plateau in GFP–AktPH translocation ( E) to interference with 5′ phosphatases and a possible suppression of the normally transient PI(3,4,5)P3
response, but rather to the ability of the probe to bind the less transient PI(3,4)P2
lipid at least equally well (Frech et al. 1997
; Kavran et al. 1998
; Gray et al. 1999
Extracellular PDGF Gradients Trigger Markedly Steeper Intracellular Gradients in 3′ Phosphoinositide Lipids
The translocation of GFP–AktPH in response to a gradient of PDGF was assessed in a temperature-controlled chamber to maintain the microscope stage at 37°C. A gradient in PDGF concentration was generated by slowly injecting a small bolus of PDGF (1.5 pmol in 5 μL) ~1 mm from the cells. With this approach, the average PDGF concentration seen by the cells increases over time, with half-maximal receptor binding occurring within minutes. The PDGF gradient, in relative terms, is estimated to be robust, maintained at 1–3% per micrometer before the saturation of PDGF receptors (Materials and Methods). Control experiments using fluorescent dextran were in agreement with these estimates (data not shown).
When PDGF was introduced by this method, GFP–AktPH translocation was consistently polarized towards the PDGF source. Strikingly, the region with the highest concentration of GFP–AktPH invariably exhibited membrane activity and the formation of lamellipodia (in six experiments, each observing two or more cells). Further, the bias in GFP–AktPH translocation and membrane extension did not depend on the orientation of the cells before stimulation with the PDGF gradient. In A, PDGF was introduced from the right, and asymmetric translocation was apparent by 2–3 min. The pronounced left-right gradient in fluorescence intensity for the cell in the upper right is shown in more detail in B, and fluorescence profiles for this cell are shown in C. Coinciding with the onset of GFP–AktPH translocation, the same cell also began spreading rapidly towards the stimulus. An analysis of the time courses of local GFP–AktPH translocation versus total cell spreading is shown in D. For later time points, the average PDGF level is sufficient to dimerize all receptors, and the translocation profile becomes more symmetrical ( C). The disparate kinetics of translocation on the right and left sides for this cell are temporally and spatially consistent with the two plateaus visible in the contact area spreading time course in D.
Figure 2 Induction of 3′ PI lipid gradients and polarized membrane spreading in response to PDGF gradients. (A) Time series of evanescent wave images showing GFP–AktPH-transfected NIH 3T3 fibroblasts responding to a transient PDGF-BB gradient at (more ...)
E shows a similar experiment in which the stimulus was directed from the left. In control experiments, pretreatment with LY294002 (100 μM) blocked both GFP–AktPH translocation as well as membrane spreading (data not shown). These results indicate that PI 3-kinase-dependent actin mobilization stimulated by PDGF is spatially focused by the localized generation of 3′ PI lipids.
Correlation of Cell Orientation and 3′ Phosphoinositide Gradients in Unstimulated Cells
An interesting feature observed for most cells was the presence of preexisting basal polarity in GFP–AktPH localization. Such basal 3′ PI gradients were observed in nearly confluent as well as in low density fibroblast cultures at 37°C, whereas basal gradients were far less prominent at room temperature. and , show basal intensity gradients in a representative experiment using nearly confluent fibroblasts. Uniform PDGF stimulation rapidly equalized the gradients on both sides of the contact area, and subsequent addition of wortmannin caused a rapid decay in fluorescence below the prestimulus level ( C) and an ablation of the basal fluorescence gradient (data not shown).
Figure 3 Basal gradients in 3′ PI lipids correlate with cell orientation. (A) Evanescent wave images of GFP–AktPH-transfected NIH 3T3 fibroblasts at 37°C, showing basal 3′ PI gradients and a more symmetrical response after uniform (more ...)
Fibroblasts plated at lower density also exhibited 3′ PI gradients in the basal state, correlating with an apparently migratory morphology. A representative example of such a cell is shown in D, for which a marked linear gradient in GFP–AktPH translocation was observed ( E). In response to uniform PDGF stimulation, two distinct spatial regimes became apparent. For the adhesion perimeter around the apparent cell body, PDGF caused the intensity to be equalized, leading to a U-shaped steady state profile similar to B. In contrast, a linear gradient was maintained in the tail. Following uniform PDGF stimulation, the slope of this tail gradient increased with time, with the fluorescence intensity at the very rear of the cell increasing only modestly compared with the cell body. These results are consistent with a spatial regulation of 3′ PI levels during cell migration.
3′ Phosphoinositide Gradients in Wounded Monolayers
Given that fibroblast migration is inhibited by contact with adjoining cells, and that 3′ PI generation appeared to be polarized away from neighboring cells (for example A), we hypothesized that the basal PI 3-kinase activity and cell orientation are related phenomena. To test this hypothesis further, we biased cell orientation by wounding a monolayer of quiescent, GFP–AktPH-transfected fibroblasts (; representative experiment selected from five separate experiments conducted at 37°C). Cells at the interface were oriented towards the acellular area by 1–2 h, after which the basal and PDGF-stimulated fluorescence patterns were assessed. All of the cells observed exhibited an apparent prestimulus 3′ PI gradient directed away from adjacent cells, with markedly brighter fluorescence associated with lamellipodia and filopodia. As in , the uniform addition of PDGF equalized the circumference fluorescence profile (in 4 of 5 wounding experiments), and addition of wortmannin rapidly erased all fluorescence gradients.
Figure 4 Demonstration of fibroblast orientation and 3′ PI gradients in wounded monolayers. Evanescent wave images showing GFP–AktPH-transfected fibroblasts at 37°C with basal gradients at the edge of a wounded monolayer. The response to (more ...)
Radial 3′ Phosphoinositide Lipid Gradients in the Cell–Surface Contact Area
The ability of 3′ PI lipids to locally enhance actin cytoskeletal activity suggests an important role in directed migration. However, this function of 3′ PIs may also regulate the size and shape of adhesive contact areas. Consistent with this hypothesis, cells with significant adhesion areas reproducibly showed radial 3′ PI gradients that increased from the center to the periphery of the contact area (for example, and ). These gradients were investigated quantitatively at room temperature (~25°C), since the lower temperature significantly suppressed basal 3′ PI gradients as well as membrane activity and spreading in response to PDGF stimulation.
A shows the translocation responses of two representative cells to uniform PDGF stimulation. After a lag period, uniform PDGF stimulation triggered a uniform fluorescence intensity increase around the periphery of the contact area, followed by an increase in fluorescence in the center as well as the periphery. Importantly, control experiments showed that larger cells expressing soluble or membrane-targeted GFP did not exhibit radial gradients, allowing several possible artifacts to be ruled out. These include variations in the membrane–substratum separation distance, edge artifacts caused by the concentration of membrane at the periphery, and extensive photobleaching. PDGF elicited neither changes in the mean fluorescence intensity nor the formation of spatial gradients in these cells ( B and data not shown).
Figure 5 PDGF-stimulated radial 3′ PI gradients in the cell contact area. (A) Representative translocation response of GFP–AktPH-transfected NIH 3T3 fibroblasts to uniformly maximal PDGF-BB stimulation at room temperature, showing radial gradients (more ...)
Surprisingly, the radial asymmetry was not caused by exclusion of PDGF from the contact zone. When fluorescent dextrans (up to 40 kD) were added in control experiments, the intensity underneath a cell was 50–70% of the fluorescence intensity elsewhere, and there was no significant lag in the intensity increase (data not shown). The slightly lower fluorescence intensity beneath the cells is an expected result from the presence of the cell within the evanescent wave-illuminated region. This indicates that the reduced concentration of 3′ PI lipids in the contact area was not caused by a lack of PDGF access, but suggests instead a segregation or feedback regulation of signaling components between the nonadherent and the adherent plasma membrane. Thus, we hypothesized that the radial pattern of 3′ PI lipids is produced by production of 3′ PI lipids in the nonadherent membrane, diffusion of 3′ PIs from the periphery into the contact area, and degradation of 3′ PI lipids within the contact area.
To further test this hypothesis, the translocation of GFP–AktPH was enhanced by coexpression with the GTPase-deficient (and therefore constitutively active with respect to signaling) G12V Ha-Ras variant. G12V Ras is expected to elicit PI 3-kinase activity independently of, and synergistically with, PDGF stimulation (Rodriguez-Viciana et al. 1994
, Rodriguez-Viciana et al. 1996
; Klinghoffer et al. 1996
). Indeed, coexpression with G12V Ras is sufficient to prelocalize most of the GFP–AktPH to the membrane as observed using confocal microscopy ( C). Using evanescent wave excitation, the average surface-proximal fluorescence transiently decreased by ~25% following a maximal dose of PDGF ( D and E). The drop in fluorescence suggests that GFP–AktPH is initially lost from the contact area due to a competition with 3′ PI lipids produced outside the contact region, supporting the hypothesis of much higher 3′ PI production in the nonadherent region.
Local Generation, Diffusion, and Degradation of 3′ Phosphoinositides
To determine how spatial gradients in 3′ PI lipids can be generated, the diffusion coefficient and degradation times of 3′ PI lipids were determined by comparing the radial patterns in GFP–AktPH translocation elicited by PDGF with a mathematical model of lipid second messenger dynamics (see http://www.jcb.org/cgi/content/full/151/6/1269/DC1). A key feature of the model is that the nonadherent portion of the plasma membrane, modeled as a hemisphere, and the circular contact area can have different second messenger production rates. The lipid diffuses laterally with observed mobility coefficient D, and it is consumed by first order reaction(s) with observed rate constant kc in both domains ( A). As a representative experiment, the spatial profiles across the width of the cell on the right in A are plotted in B (the cell on the left exhibited similar behavior). The profile before stimulation was flat, and, during the first stages of the translocation response, the intensity in the center of the contact area actually decreased. A smooth, U-shaped profile was established within 45 s of stimulation, attaining a steady state by 2–3 min.
C shows model calculations assuming negligible second messenger production in the contact area. The other parameters were adjusted to yield agreement with B (see Materials and Methods), capturing all quantitative aspects of the experimental profiles. For example, the fluorescence decrease in the center of the profile is attributed to the depletion of GFP–AktPH from the cytosol, with diffusion being too slow to compensate for early time points. Of particular interest are the estimated values of the 3′ PI diffusion coefficient D (~0.5 μm2
/s) and specific turnover rate kc
). The estimated diffusion coefficient is in approximate agreement with mobility studies of membrane lipid probes and Ras, which is lipid-anchored (Schlessinger et al. 1977
; Niv et al. 1999
). To estimate the degradation rate constant independently, GFP–AktPH expressing cells were stimulated with PDGF for 5 min, at which time 250 μM LY294002 was added to rapidly inhibit further 3′ PI generation. The mean decay constant of the fluorescence intensity was determined to be 1.0 ± 0.4 min−1
( D), in agreement with the model estimate. Also estimated was the parameter σ, the fold-increase in fluorescence intensity when a probe molecule is membrane-bound versus freely diffusing in the cytosol. Both the model and the experimental data in C determined this quantity to be over an order of magnitude.
Though the analysis of the translocation kinetics at 37°C was confounded by changes in the shape of the contact area, the data was consistent with a moderately faster turnover rate (~1.5 min−1) than at room temperature ( and , and data not shown). With this estimated turnover rate, the steady-state fluorescence profiles were consistent with a lipid diffusion coefficient not significantly affected by temperature in this range.
We also simulated the alternative situation where consumption of the second messenger is fast relative to diffusion, with the second messenger concentration at the center of the contact area being compensated by a nonzero generation rate ( E). In this case, the transition from the periphery to the center is too steep, and the model fails to predict a decrease in fluorescence for early time points. Also giving poor agreement with experiment was the situation where the second messenger generation rates are equal in the two domains, but the turnover rate is higher in the contact area (not shown). Thus, the observed radial variation in PDGF-stimulated fluorescence can be explained simply by lipid diffusion into the contact area from the nonadherent part of the cell.